Long-term propagation of serum hepatitis C virus (HCV) with production of enveloped HCV particles in human HepaRG hepatocytes

Abstract

HepaRG human liver progenitor cells exhibit morphology and functionality of adult hepatocytes. We investigated the susceptibility of HepaRG hepatocytes to in vitro infection with serum-derived hepatitis C virus (HCV) particles (HCVsp) and the potential neutralizing activity of the E1E2-specific monoclonal antibody (mAb) D32.10. The infection was performed using HCVsp when the cells actively divided at day 3 postplating. HCV RNA, E1E2, and core antigens were quantified in HCV particles recovered from culture supernatants of differentiated cells for up to 66 days. The density distributions of particles were analyzed on iodixanol or sucrose gradients. Electron microscopy (EM) and immune-EM studies were performed for ultrastructural analysis of cells and localization of HCV E1E2 proteins in thin sections. HCV infection of HepaRG cells was documented by increasing production of E1E2-core-RNA(1) HCV particles from day 21 to day 63. Infectious particles sedimented between 1.06 and 1.12 g/mL in iodixanol gradients. E1E2 and core antigens were expressed in 50% of HCV-infected cells at day 31. The D32.10 mAb strongly inhibited HCV RNA production in HepaRG culture supernatants. Infected HepaRG cells frozen at day 56 were reseeded at low density. After only 1-3 subcultures and induction of a cell differentiation process the HepaRG cells produced high titer HCV RNA and thus showed to be sustainably infected. Apolipoprotein B-associated empty E1E2 and complete HCV particles were secreted. Characteristic virus-induced intracellular membrane changes and E1E2 protein-association to vesicles were observed. Conclusion: HepaRG progenitor cells permit HCVsp infection. Differentiated HepaRG cells support longterm production of infectious lipoprotein-associated enveloped HCV particles. The E1E2-specific D32.10 mAb neutralizes the infection and this cellular model could be used as a surrogate infection system for the screening of entry inhibitors. (HEPATOLOGY 2011;54:406-417) H epatitis C virus (HCV) infection is a major health problem worldwide because at least 70% of infections persist and cause chronic hepatitis, which may progress to liver cirrhosis and hepatocellular carcinoma. 1 The lack of robust cell culture and small animal models remain stumbling blocks to

Experimental models of hepatitis B and C — new insights and progress

“…An additional cell line has proven to be useful for studies of both HBV 81 and HCV 82 : the HepaRG cell line. This cell line was developed from cells obtained from a resection of tumour from an HCV-infected individual with liver cancer, although the infection was not sustained in vitro after isolation of these cells.…”

“…Unfortunately, the use of polyethylene glycol is needed to facilitate viral entry by increasing interactions between the HBV virion and the cell membrane but the overall infection rate is low with minimal cell-to-cell virus spread 85 . Later, investigators were able to use this cell line as a substrate for HCV infection 82 . In this study 82 , the investigators were able to utilize serum-derived HCV genotype 3 to validate susceptibility to HCV infection 55 .…”

“…Later, investigators were able to use this cell line as a substrate for HCV infection 82 . In this study 82 , the investigators were able to utilize serum-derived HCV genotype 3 to validate susceptibility to HCV infection 55 .…”

Cell Culture Systems for Hepatitis C Virus

“…A human liver progenitor cell line, named HepaRG, can be differentiated into hep atocytes with specific cell culture conditions and has been widely used for HBV infection experiments. It could now be shown that these cells are also susceptible to serum-derived HCV particles and support long-term production of viral particles, albeit at very low levels (Ndongo-Thiam, Berthillon et al 2011). Primary human hepatocytes (PHH) provide the closest in vitro model for the natural host cell of HCV.…”

Hepatocyte-like cells differentiated from human induced pluripotent stem cells (iHLCs) are permissive to hepatitis C virus (HCV) infection: HCV study gets personal

“…Recently, HepaRG progenitor cells were shown to be capable of differentiating into hepatocytes and biliary epithelial cells, depending on culture conditions [3]. These progenitors were used as a surrogate for primary human hepatocytes [4], and showed to be susceptible to in vitro infection with serum-derived HCV (genotype 3) and able to support long-term production of infectious lipoprotein-associated enveloped HCV particles [5]. Taking these findings a step further, the model described by Schwartz, Trehan et al offers the possibility to study genetic defects that impact HCV infection of human iPS-derived hepatocyte-like cells (iHLCs) [6].…”